Vertebrate forelimbs (arms) and hindlimbs (legs) are homologous tissues, and share many patterning programs for development. Surprisingly, however, recent studies suggest that the upstream regulatory programs controlling common genetic programs for their development seem to be genetically distinct. Identifying limb-type specific, distinct upstream regulatory mechanisms that control common processes, and unveiling how these programs differ, are important unanswered questions in developmental biology. This proposal will address these questions by elucidating the mechanisms of spatial activation of the Sonic hedgehog (Shh) gene specifically in hindlimb buds. During early stages of the development of both fore- and hindlimb buds, Shh expression is induced in the posterior mesenchyme through a cis-element directly regulated by HAND2 and HOX proteins. However, upstream regulations of Hand2 seem to be different in fore- and hindlimb buds. It is reported that in Hox9 quadruple knockout (Hox-a9, b9, c9, d9), Hand2 and Shh expression were not activated in forelimb buds, while expression of Hand2 and Shh in hindlimb buds was normal. It was envisaged that a similar scenario involving different Hox paralogs could control Hand2 and Shh in early hindlimb buds. However, no Hox genes that regulate the early expression of Hand2 and Shh in hindlimb buds have been identified. Thus, the hindlimb bud- specific mechanism acting upstream of the Hand2-Shh pathway remains elusive. Preliminary data for this proposal, obtained by conditional knockout (cKO) of Islet1 (Isl1) and Gata6, suggests that these two genes are upstream of Hand2-dependent Shh induction specifically in hindlimb buds. Based on observation and further analysis, the specific hypothesis of the proposed project is that spatial restriction of Isl1 to the posterior region of the hindlimb-forming area by Gata6 triggers the Hand2-Shh pathway in the posterior region of the hindlimb bud. Aim 1 will determine whether negative regulation by Gata6 restricts Isl1 for hindlimb-specific activation of the Hand2-Shh pathway. Virus-mediated ectopic expression analyses of Isl1 and Gata6, followed by examination of marker genes/proteins, will be carried out. Double KO of Gata6 and Isl1 will be performed to determine their relationship in Shh activation in hindlimb buds. Aim 2 will determine the mechanism by which Gata6 negatively regulates Isl1 through its cis-regulatory element. In vitro DNA binding assay, luciferase reporter assay to test transcriptional regulation, in vivo chromatin immunoprecipitation assay to test in vivo binding and mouse embryo LacZ reporter assay will be performed. Combined, this proposal will determine that different transcription factors (Hox9s in fore- and Isl1 in hindlimb buds) act as regulators of a common Hand2-Shh pathway in two homologous tissues. Moreover, the proposal will identify a novel upstream regulation system by Gata6 that leads to correct spatial activation of Shh in a hindlimb specific manner.